Topical microbicides, designed for genital or rectal administration, are needed to prevent HIV infection. Further rational development of microbicides should build on prior clinical experiences demonstrating a clear benefit of systemic combination antiretroviral therapy and the unexpected toxicity of topical agents. This Program will systematically study local virologic and immunologic effects of PRO 2000, with the establishment of assays and models that might better predict outcome. The sound principle of combination therapy will be applied to the analysis of topicals with in vitro studies aimed at identifying the most promising combinations. PRO 2000, a naphthalene sulfonic acid, is active against a wide range of HIV isolates in vitro and inhibits a major HIV cofactor, the herpes simplex virus (HSV). The drug acts by binding viral envelope glycoproteins and preventing entry. Vaginal gel formulations containing up to 4 percent PRO 2000 have been shown to be safe and well tolerated in two Phase I clinical trials. A combination of PRO 2000 with a second microbicide that targets a different step in the HIV life cycle should have distinct advantages over either drug alone, including enhanced efficacy, reduced mucosal inflammation and an expanded spectrum of activity against other sexually transmitted pathogens. An iterative approach toward identifying the best combination of PRO 2000 with a second microbicide is proposed. PRO 2000 combined with PMPA (tenovofir), a nucleotide analogue, and UC781, a non-nucleoside reverse transcriptase inhibitor (RTI) will be tested. These compounds are being expeditiously advanced as independent candidates for microbicide development. PMPA and UC781 inhibit HIV reverse transcription, a step after viral entry. A multitargeted combination is likely to be more effective because PRO 2000 blocks cell entry and the RTIs inhibit virus that successfully evades PRO 2000. Novel candidate virucidal agents in preclinical development, including bile salts and amphiphilic polymers, will also be evaluated. In three interrelated projects, the antiviral activity and inflammatory effects will be examined advancing combinations from cell and explant culture systems to animal models and pilot clinical studies. Project 1 will focus on identifying compounds that act additively or synergistically against clinical isolates of HIV using primary cells and explant cultures. Project 2 will evaluate the impact of microbicide combinations on a dual infection model of HSV and HIV using human explant cultures. In murine genital and rectal models, inflammatory effects will be assessed as well as the activity against HSV-2. In Project 3, three pilot human clinical trials will be conducted to examine the effects of PRO 2000 on genital tract HIV and on mucosal immunity in both HIV-infected and uninfected individuals. The proposed comprehensive preclinical evaluation of combinations and clinical evaluation of PRO 2000 will lay the groundwork for future clinical trials with optimal combinations.